Canine and feline oocyte lipid profile by Matrix-Assisted Desorption Ionization time-of-flight Mass Spectrometry (MALDI-TOF MS)

OBJECTIVES AND METHODS. Canine and Feline oocytes present an elevate cryosensibility, mostly due to their high lipid content (1). It is believed that the presence of intracellular lipid droplets could be responsible for uneven intracellular ice formation which could affect the freezing-thawing process (2). Studies on the lipid composition of oocytes from these species may contribute with the improvement of the cryopreservation process as well as to the advancement of in vitro culture conditions. Thus, the aim of this work is to apply the technique of matrix-assisted desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) to obtain the lipid profiles of single canine and feline oocytes and compare with the lipid profiles obtained for the bovine oocytes, a species commonly used for cryopreservation studies. Ovaries were collected from three diestrous bitches (various breeds, age 1-7 y) and one queen by routine ovariohysterectomy and sliced in PBS-PVA to release the cumulus-oocyte complexes (COCs). Oocytes were graded according to morphological characteristics and only grade I COCs were selected and stored in PBS solution at -20C until analysis. For MALDI-TOF MS analysis, samples were washed in H2O/MeOH 1:1 (v/v) and placed (1 to 3 oocytes/spot) in the MALDI target plate. Samples were then covered with 2.5 dihydroxybenzoic acid (DHB) as the organic matrix. A Synapt HDMS (Waters Corp. Milford, MA, USA) equipped with a MALDI source was used for the experiments. All mass spectra were manually collected for around 1 min in the positive ion mode, at the m/z range of 700-1000. Mass spectra collected from individual embryos were processed using the software MassLynx 4.1 (Waters Corp. Milford, MA, USA). RESULTS. This is the first report about lipid fingerprint of canine and feline oocytes by MALDI-TOF MS. The technique has been already reported for bovine oocytes and embryos, as well as human and sheep oocytes (3). Besides the analysis workflow simplicity (no lipid extraction), intact complex lipid species such as sphingomyelins (SM), phosphocholines (PC) and triacylgycerols (TAG) can be detected because lipids are easily ionizable by MALDI using DHB as the matrix. By gas chromatography (GC), traditionally used for lipid analysis in gametes and embryos, only fatty acyl residues can be detected and structural information of lipids is lost. Mass spectra of bitch and queen oocytes were similar and characterized mainly by intense ions correspondent to the m/z values of PC containing 34 carbons (of m/z 780.7 to 786.7) and 36 carbons (of m/z 808.7 to 812.7), but mainly PC (34:1) of m/z 782.8 (sodiated PC 34:1), m/z 723.7 (fragment of PC 34:1) and 760.8 (protonated PC 34:1), which is the main phospholipid present in the cellular membrane and this fact has been also observed for bovine oocytes (3). Regarding the TAG species, m/z values correspondent to species containing 52 and 54 carbons were more prominent in canine and especially in feline oocytes, compared to the bovine (3). CONCLUSIONS. Lipid attributions described herein must be confirmed by MS/MS experiments, but our data indicate that MALDI-TOF MS is able to detect the presence and the chemical structure (number of carbons present in the fatty acyl residues and unsaturations) of PC and TAG species in canine and feline oocytes. We envisage MALDI-TOF MS will become a relevant tool in studies aiming at improving canine and feline oocyte cryopreservation success. (1) -Luvoni GC, Pellizzari P, Battocchio M. Effects of slow and ultrarapid freezing on morphology and resumption of meiosis in immature cat oocytes. J Reprod Fertil Suppl 1997; 51: 93-98. (2) Luvoni GC. Gamete cryopreservation in the domestic cat. Theriogenology 2006; 66: 101-11. (3) Ferreira CR, Saraiva SH, Catharino RR, Garcia JS, Gozzo FC , Sanvido GB, Santos LFA, Lo Turco EG, Pontes JHF, Basso AC, Bertolla RP, Sartori R, Guardieiro MM , Perecin F , Meirelles FV , Sangalli JR, Eberlin MN. Single embryo and oocyte lipid fingerprinting by mass spectrometry. Journal of lipid research 2010; 51: 1218-1227.